Project Summary and Abstract The RAS gene family is among the most commonly mutated genes in human cancer with nearly 30% containing a mutation in a RAS gene. The three RAS genes (KRAS, NRAS, and HRAS) play a central role in cell signal transduction as GTPase switches, and mutations in RAS proteins lead to constitutive activity resulting in oncogenic transformation. Despite years of intensive research, little progress has been made in successfully targeting mutant RAS clinically, making the identification of novel RAS interactors a potentially attractive strategy for the development of targeted therapeutics. Recent studies have identified an interaction between the N terminal domain of Argonaute 2 (AGO2), a core component of RNA-induced silencing complex (RISC), and the Switch II domain of KRAS across both wild-type (WT) and mutant KRAS expressing cell lines. Furthermore, this interaction was functionally important to KRAS-mediated growth. Stable knockdown of AGO2 in KRAS dependent cells led to a decrease in KRAS protein expression with a subsequent decrease in cellular proliferation. In addition, interaction with mutant KRAS inhibits the RNAi function of AGO2 by preventing microRNA unwinding. This study will further investigate the role of growth factor regulation of AGO2-RAS in both normal physiology and cancer biology. EGFR activation was demonstrated to attenuate the interaction of AGO2 and WT RAS in normal cells by AGO2 phosphorylation, but mutant KRAS was resistant to growth factor regulation. Despite the potential regulatory role for growth factor signaling in AGO2-RAS biology, the precise mechanism and functional role of this regulation is unknown. Here we propose to study the AGO2-RAS-EGFR axis through the following Aims: Specific Aim 1: EGFR-mediated phosphorylation of AGO2 alters RAS-AGO2 interaction that may lead to changes in RAS activity and cellular localization. Here, we will assess EGFR regulation of AGO2 phosphorylation, RAS localization, and RAS-GTP loading in normal physiology and cancer biology. Specific Aim 2: Given that mutant KRAS is known to inhibit AGO2 miRNA unwinding, iCLIP-seq will be performed in the presence of AGO2-phosphomutants and/or mutant KRAS to determine changes in AGO2 miRNA processing and targeting following growth factor stimulation. We will also determine the effect of growth factor stimulation on global AGO2 miRNA processing, targeting, and gene regulation. Specific Aim 3: Preliminary studies demonstrated that loss of AGO2 leads to an increase in pEGFR and RAS activity despite serum starvation, suggesting an important bidirectional signaling and crosstalk between AGO2, RAS and EGFR. We will characterize the AGO2-RAS-EGFR axis and its role in cell survival and growth. In summary, the results of this project are expected to improve understanding of growth factor regulation of AGO2-RAS in cancer biology and may provide clinical targets to treat mutant RAS-driven cancers.